Novel ibuprofen prodrugs with improved pharmacokinetics and non-ulcerogenic potential

Article abstract of DOI:10.1007/s00044-013-0639-8

In the present study, we evaluated the anti-inflammatory activity with pharmacokinetic, ulcerogenic properties of various synthesized prodrugs of ibuprofen in experimental animals. Prodrugs 2, 6, 9, 10, 12, and 14 were found to possess significant anti-inflammatory activity with almost non-ulcerogenic potential than standard drug ibuprofen 1a in both normal and inflammation-induced rats. Metabolic stability of prodrugs 2, 6, 9, 10, 12, and 14 were also studied in rat liver microsomes and oral bioavailability was determined by estimating area under curve (AUC) and plasma concentration of these prodrugs at various time intervals. The experimental findings elicited higher AUC and plasma concentration at 1 and 2 h indicating improved oral bioavailability as compared to parent ibuprofen. These prodrugs are found to have least gastric ulceration with retain anti-inflammatory activity observed in experimental animals. Therefore, present experimental findings demonstrated significant improvement of various pharmacokinetic properties with least ulcerogenic potential of ester prodrugs of ibuprofen an anti-inflammatory agent

Full text of DOI:10.1007/s00044-013-0639-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0639-8
ORIGINAL RESEARCH
Novel ibuprofen prodrugs with improved pharmacokinetics
and non-ulcerogenic potential
•
Valmik D. Dhakane Hemant V. Chavan
•
•
Vishnu N. Thakare Laxman K. Adsul
•
•
Sadanand N. Shringare Babasaheb P. Bandgar
Received: 15 March 2013 / Accepted: 20 May 2013
Ó Springer Science+Business Media New York 2013
Abstract In the present study, we evaluated the anti-
inflammatory activity with pharmacokinetic, ulcerogenic
properties of various synthesized prodrugs of ibuprofen in
experimental animals. Prodrugs 2, 6, 9, 10, 12, and 14 were
found to possess significant anti-inflammatory activity with
almost non-ulcerogenic potential than standard drug ibu-
profen 1a in both normal and inflammation-induced rats.
Metabolic stability of prodrugs 2, 6, 9, 10, 12, and 14 were
also studied in rat liver microsomes and oral bioavailability
was determined by estimating area under curve (AUC) and
plasma concentration of these prodrugs at various time
intervals. The experimental findings elicited higher AUC
and plasma concentration at 1 and 2 h indicating improved
oral bioavailability as compared to parent ibuprofen. These
prodrugs are found to have least gastric ulceration with
retain anti-inflammatory activity observed in experimental
animals. Therefore, present experimental findings demon-
strated significant improvement of various pharmacokinetic
properties with least ulcerogenic potential of ester prodrugs
of ibuprofen an anti-inflammatory agent
Keywords Prodrug ꢀ Ibuprofen ꢀ
Anti-inflammatory activity ꢀ Non-ulcerogenic ꢀ
Pharmacokinetic properties
Abbreviations
NSAID
Nonsteroidal anti-inflammatory drug
Tris(hydroxymethyl)aminomethane
hydrochloride
Tris–HCl
NADPH
HPLC
GC
Nicotinamide adenine dinucleotide phosphate
High performance liquid chromatography
Gas chromatography
RLM
RP
Rat liver microsomes
Rat plasma
API
Active pharmaceutical ingredient
Ulcer index
UI
GI
Gastrointestinal
DMAc
PTSA
PXRD
RT
Dimethyl acetamide
p-Toluene sulfonic acid
Powder X-ray diffraction
Room temperature
TMG
1,1,3,3-Tetramethyl guanidine
18 Crown 6 1,4,7,10,13,16-Hexaoxacyclooctadecane
EP
European pharmacopoeia
Aromatic
Ar
V. D. Dhakane ꢀ H. V. Chavan ꢀ L. K. Adsul ꢀ
S. N. Shringare ꢀ B. P. Bandgar (&)
BDL
Below detection limit
Medicinal Chemistry Research Laboratory, School of Chemical
Sciences, Solapur University, Solapur 413 255,
Maharashtra, India
Introduction
e-mail: bandgar_bp@yahoo.com
H. V. Chavan
e-mail: hemantchavan.sus@rediffmail.com
Ibuprofen, a well-known nonsteroidal anti-inflammatory
drug (NSAIDs) most widely used and represents a mainstay
in the therapy of acute and chronic pain and inflammation
viz. osteoarthritis and rheumatoid arthritis (Coruzzi et al.,
2007; Kawail et al., 2005; Klasser and Epstein, 2005). The
V. N. Thakare
Department of Pharmacology, Sinhgad Institute
of Pharmaceutical Sciences, Kusgaon (Bk),
Lonavala 410 401, Maharashtra, India
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Med Chem Res
anti-inflammatory activity of ibuprofen is mainly due to its
inhibitory effect on prostaglandin synthase (cyclooxygen-
ase) which is involved in the formation of various inflam-
matory prostaglandins. However, the use of ibuprofen is
frequently associated with an adverse effect, in particular its
gastritis associated with ulceration, which are related to
inhibiting prostaglandin synthesis. These adverse effects are
likely due to the presence of carboxylic group and inhibition
of protective cyclooxygenase-1(COX-1) enzyme in stomach
(Rainsford, 1988; Bhosale et al., 2006). Inhibition of
cyclooxygenase-2 (COX-2) enzyme in desirable with ibu-
profen for its anti-inflammatory activity is concern; how-
ever, simultaneously the COX-1 inhibition is documented
with ibuprofen which is undesired. Selective inhibition of
COX-2 with coxibs (roficoxib, valdecoxib) are of great
importance in inflammation. However, their serious cardio-
vascular effects (stroke) observed from clinical studies and
post marketing surveillance, forcing the pharma companies
to withdraw these coxibs from the market (Bresalier et al.,
2005; Nussmeier et al., 2005). Precondition for the prodrugs
possesses good pharmacological (anti-inflammatory) effect,
differentiated pharmacokinetic property (appropriate aque-
ous solubility and lipophilicity) and no ulcerogenic action.
Various ester prodrugs of NSAIDs were found to possess
potent anti-inflammatory activity with lesser ulcerogenic
property on gastrointestinal (GI) mucosa Perioli et al., 2004;
Halen et al., 2009; Shanbhag et al., 1992; Chen et al., 2009;
Borhade et al., 2012.
anti-inflammatory activity (Huang et al., 2011). However, the
aim of synthesis of prodrugs of NSAIDs is to prevent the
adverse effect by protecting GI damage and this effect can be
masked byproducing inert promoeities. Because there may be
a chance of other side effect that might be associated with NO
on the other organ system apart from GI.
Based on the scientific reports on ibuprofen prodrugs
which are of great interest of reducing GI toxicity and
improved biopharmaceutical properties further experiments
are required to design and identify prodrugs of ibuprofen,
which would be suitable for clinical practice in terms of
stability, metabolism, toxicology and side effects. Ibupro-
fen is presently marketed as a racemic mixture of active
substance for its anti-inflammatory and analgesic actions.
Therefore, racemic mixture of ibuprofen is taken for the
preparation of prodrugs.
The present research work is an attempt to develop pro-
drugs of ibuprofen 1–15, using novel promoeities 2a–o
having high in vivo enzymatic conversion rate, anti-inflam-
matory, physicochemical and non-ulcerogenic properties.
Experimental section
Materials and methods
All reactions were monitored by thin layer chromatography
(TLC) with 0.2 mm Merck silica gel F254 plates and GC.
NMR spectra were recorded on Bruker DRX FT NMR at
400 MHz spectrometer in CDCl3 using TMS as internal
standard (chemical shifts are expressed as d values relative
to TMS as internal standard). IR spectra were recorded on a
FTIR Perkin-Elmer model RXI spectrophotometer. Mass
spectra were recorded on an Agilent GCMS-5975C VL
MSD spectrophotometer. HPLC analysis was performed
using a Shimadzu LC 2010 CHT with UV detector. All
prodrugs 1–15 were analyzed by HPLC, and their purity
was confirmed to be excess of 98 %. GC analysis was
performed on Shimadzu GC-2014 instrument, and purity of
promoieties 2a–o was confirmed to be in excess of 97 %.
In vivo study was performed using experimental protocols
reviewed and approved by the Institutional Animal Ethics
Committee (SIPS/IAEC/2011-12/17) and conform to the
Indian National Science Academy Guidelines for the use
and care of experimental animals in research. The animal
house Registration No. with Government of India is 962/c/
06/CPCSEA.
The majority of NSAID prodrugs have been produced
by masking of carboxylic functional group. The esters have
dominated prodrug research due to their ideal characteristic
of exhibiting reasonable in vitro chemical stability,
allowing them to formulate with desired t1/2. Because of
its ability to functions as esterase substrates, esters are
suitably liable in vivo condition (Bundgaard, 1986, 1989).
Therefore, by the use of prodrug approach, one can mask
the carboxylic acid functional group of NSAID, which on
hydrolysis, an active parent drug to be released in vivo for
its anti-inflammatory activity along with reduction in
ulcerogenic property (Gairola et al., 2005; Wallace, 1994;
Kim et al., 2006).
Toxicological and pharmacological profiles of ibuprofen
guiacol ester (Cioli et al., 1980), synthesized ester prodrugs
using N-hydroxymethylsuccinimide and N-hydroxymeth-
ylisatin as promoeities to reduce their GI toxicity and
improve bioavailability (Mahfouz et al., 1999), 2-formyl-
phenyl esters (Abordo et al., 1998) was studied. However,
their release characteristic is found to exhibits variation.
And most of the prodrugs of NSAIDs reported in the lit-
eratures have poor or limited hydro solubility (Nielsen and
Bundgaard, 1988; Shaaya et al., 2003).
General procedure for the preparation of prodrugs
(1–15)
The various studies onprodrugssuggested that, nitric oxide
(NO) release elicits cytoprotective activity and retained
Ibuprofen (2 g, 9.67 mmol) was dissolved in dimethyl acet-
amide (25 mL) at room temperature (25 °C). To this solution,
123

Med Chem Res
2-(Cyclohexyloxy)-2-oxoethyl-2-(4-isobutylphenyl)propan-
oate (4) Chromatographic purity (HPLC) 99.91 %; bp [
300 8C; MS (ESI?) m/z = 346 [M?]; IR (KBr, cm-1): 2938,
2865, 1746, 1614, 1514, 1455, 1150, 845; 1H NMR (CDCl3,
400 MHz) d 0.86 (d, J = 6.4 Hz, 6H, CH(CH3)2), 1.19–1.34
(m, 6H, 39CH2), 1.60–1.71 (m, 4H, 29CH2), 1.41 (d,
J = 7.2 Hz, 3H, ArCHCH3), 1.80 (m, 1H, CH(CH3)2), 2.42
(d, J = 7.2 Hz, 2H, ArCH2CH), 3.85 (q, J = 7.2 Hz, 1H,
ArCHCH3), 4.61 (s, 2H, OCH2CO), 4.69 (m, 1H, COOCH),
7.12 (d, J = 8 Hz, 2H, ArH), 7.20 (d, J = 8 Hz, 2H, ArH).
potassium carbonate (4 g, 28.9 mmol) under nitrogen atmo-
sphere was added. The reaction mass was stirred for 20 min at
room temperature (25 °C). Then, to the above reaction mass,
promoieties 2a–2o (8.7 mmol) was added under stirring and
maintained the reaction under stirring for 4–5 h at room
temperature. The reaction progress was monitored by TLC
and GC. After completion, the reaction mass was added under
stirring into the mixture of ethyl acetate (60 mL) and water
(100 mL). The organic layer was separated and aqueous layer
extracted with ethyl acetate twice (60 mL 9 2). Combined
organic layer washed with 20 % solution of sodium bicar-
bonate (100 mL 9 2) followed by brine wash (10 % NaCl,
100 mL). Organic layer was dried over anhydrous sodium
sulfate, distilled off the solvent under vacuum and desired
product collected as viscous oil.
2-(2-Ethylbutoxy)-2-oxoethyl-2-(4-isobutylphenyl)propan-
oate (5) Chromatographic purity (HPLC) 99.78 %; bp 291
8C; MS (ESI?) m/z = 348 [M?]; IR (KBr, cm-1): 2962,
2875, 1747, 1513, 1463, 1152, 847; 1H NMR (CDCl3,
400 MHz) d 0.84–0.88 (m, 12H, 49CH3), 1.23–1.34 (m, 3H,
CHCH2CH3/CHCH2CH3); 1.50 (qu, 2H, CHCH2CH3), 1.41
(d, J = 7.2 Hz, 3H, ArCHCH3), 1.81 (m, 1H, CH(CH3)2),
2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.85 (q, J = 7.2 Hz,
1H, ArCHCH3), 4.05(t, J = 6.4 Hz, 2H, COOCH2CH), 4.64
(s, 2H, OCH2CO), 7.11 (d, J = 8 Hz, 2H, ArH), 7.20 (d,
J = 8 Hz, 2H, ArH).
Spectral data of synthesized compounds
Ethyl 2-(2-(4-isobutylphenyl)propanoyloxy)-3-oxobutano-
ate (1) Chromatographic purity (HPLC) 99.78 %; bp 298
8C; MS (ESI?) m/z = 334 [M?]; IR (KBr, cm-1): 2957,
1
2871, 1745, 1633, 1609, 1514, 1369, 1156, 852; H NMR
(CDCl3, 400 MHz) d 0.89 (d, J = 6.8 Hz, 6H, CH(CH3)2),
1.23 (t, J = 7.2 Hz, 3H, OCH2CH3); 1.57 (m, 3H,
ArCHCH3), 1.84 (m, 1H, CH(CH3)2), 2.08 (s, 3H, COCH3),
2.45 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.90 (q, J = 7.2 Hz,
2H, OCH2CH3), 4.24 (q, J = 7.2 Hz, 1H, ArCHCH3), 5.43
(s, 1H, OCHCO), 7.10 (d, J = 8 Hz, 2H, ArH), 7.25 (d,
J = 8 Hz, 2H, ArH).
2-(2-Methoxyethoxy)-2-oxoethyl-2-(4-isobutylphenyl)pro-
panoate (6) Chromatographic purity (HPLC) 99.77 %; bp
288 8C; MS (ESI?) m/z = 322 [M?]; IR (KBr, cm-1): 2955,
2870, 1746, 1513, 1458, 1157, 847; 1H NMR (CDCl3,
400 MHz) d 0.88 (d, J = 6.6 Hz, 6H, CH(CH3)2), 1.54 (d,
J = 7.2 Hz, 3H, ArCHCH3), 1.84 (m, 1H, CH(CH3)2), 2.45
(d, J = 7.2 Hz, 2H, ArCH2CH), 3.36 (s, 3H, OCH3), 3.56
(m, 2H, CH2OCH3), 3.81 (q, J = 7.2 Hz, 1H, ArCHCH3),
4.28 (m, 2H, COOCH2), 4.59 (s, 2H, OCH2CO), 7.11 (d,
J = 8 Hz, 2H, ArH), 7.22 (d, J = 8 Hz, 2H, ArH).
4-Acetoxybutyl-2-(4-isobutylphenyl)propanoate (2) Chro
matographic purity (HPLC) 99.60 %; bp [ 300 8C; MS
(ESI?) m/z = 320 [M?]; IR (KBr, cm-1): 2945, 2870, 1743,
1614, 1515, 1460, 1151, 843; 1H NMR (CDCl3, 400 MHz) d
0.88 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.49 (d, J = 7.2 Hz,
3H, ArCHCH3), 1.54–1.68 (m, 4H, CH2CH2CH2O), 1.84 (m,
1H, CH(CH3)2), 2.02 (s, 3H, COCH3), 2.45 (d, J = 7.2 Hz,
2H, ArCH2CH), 3.69 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.01
(t, J = 6 Hz, 2H, CH2COOCH3), 4.08 (t, J = 6 Hz, 2H,
CH2OCOCH), 7.09 (d, J = 8 Hz, 2H, ArH), 7.20 (d,
J = 8 Hz, 2H, ArH).
2-Isopropoxy-2-oxoethyl-2-(4-isobutylphenyl)propanoate
(7) Chromatographic purity (HPLC) 98.34 %; bp 266 8C;
MS (ESI?) m/z = 306 [M?]; IR (KBr, cm-1): 2976, 2870,
1741, 1516, 1462, 1373, 1105, 845; 1H NMR (CDCl3,
400 MHz) d 0.84 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.14 (d,
J = 6.4 Hz, 6H, OCH(CH3)2), 1.41 (d, J = 7.2 Hz, 3H,
ArCHCH3), 1.81 (m, 1H, CH2CH(CH3)2), 2.42 (d,
J = 7.2 Hz, 2H, ArCH2CH), 3.85 (q, J = 7.2 Hz, 1H,
ArCHCH3), 4.59 (s, 2H, OCH2CO), 4.91 (sept, J = 6.4 Hz,
1H, OCH(CH3)2), 7.12 (d, J = 8 Hz, 2H, ArH), 7.23 (d,
J = 8 Hz, 2H, ArH).
1-Methoxy-1-oxopropan-2-yl-2-(4-isobutylphenyl)propan-
oate (3) Chromatographic purity (HPLC) 99.69 %; bp
276 8C; MS (ESI?) m/z = 292 [M?]; IR (KBr, cm-1):
1
2955, 2870, 1744, 1614, 1514, 1455, 1160, 1101, 848; H
2-Butoxy-2-oxoethyl-2-(4-isobutylphenyl)propanoate (8) Chro
matographic purity (HPLC) 99.67 %; bp 275 8C; MS (ESI?)
m/z = 320 [M?]; IR (KBr, cm-1): 2958, 2871, 1747, 1514,
1458, 1153, 846; 1H NMR (CDCl3, 400 MHz) d 0.80–0.86
(m, 11H, 39CH3, CH2CH3), 1.25 (qu, J = 6.8 Hz, 2H,
OCH2CH2), 1.41 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.80 (m,
1H, CH(CH3)2), 2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.85
NMR (CDCl3, 400 MHz) d 0.90 (d, J = 6.4 Hz, 6H,
CH(CH3)2), 1.42 (d, J = 7.2 Hz, 1H, OCHCH3), 1.53 (d,
J = 7.2 Hz, 3H, ArCHCH3), 1.84 (m, 1H, CH(CH3)2),
2.45 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.62 (s, 3H,
COOCH3), 3.79 (q, J = 7.2 Hz, 1H, ArCHCH3), 5.08 (q,
J = 7.2 Hz, 1H, OCHCH3), 7.10 (d, J = 8 Hz, 2H, ArH),
7.22 (d, J = 8 Hz, 2H, ArH).
123

Med Chem Res
1
2937, 2870, 1740, 1513, 1465, 1376, 1103, 847; H NMR
(q, J = 7.2 Hz, 1H, ArCHCH3), 3.98 (m, 2H, OCH2CH2),
4.65 (s, 2H, OCH2CO), 7.11 (d, J = 8 Hz, 2H, ArH), 7.20 (d,
J = 8 Hz, 2H, ArH).
(CDCl3, 400 MHz) d 0.81–0.94 (m, 9H, 39CH3), 1.11 (d,
J = 6.4 Hz, 3H, CH3CH), 1.20 (d, J = 6.4 Hz, 3H,
CH3CH), 1.51 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.55–1.86
(m, 3H, CH(CH3)2/CH3CH2CH), 2.43 (d, J = 7.2 Hz,
ArCH2CH), 3.79 (m, 1H, ArCHCH3), 4.81–4.88 (m, 1H,
OCH(CH3)2), 4.95–5.07 (m, 1H, OCHCO), 7.07–7.10 (m,
2H, ArH), 7.22–7.26 (m, 2H, ArH).
2-(Benzyloxy)-2-oxoethyl-2-(4-isobutylphenyl)propanoate
(9) Chromatographic purity (HPLC) 98.81 %; bp [ 300
8C; MS (ESI?) m/z = 354 [M?]; IR (KBr, cm-1): 2954,
1
2848, 1747, 1608, 1514, 1455, 1150, 847, 750, 698; H
NMR (CDCl3, 400 MHz) d 0.85–0.90 (m, 6H, CH(CH3)2),
1.52 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.84 (m, 1H,
CH(CH3)2), 2.43 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.82 (q,
J = 7.2 Hz, 1H, ArCHCH3), 4.54 (s, 2H, COOCH2CO),
4.05–4.10 (m, 2H, OCH2Ph), 7.11 (d, J = 8 Hz, 2H, ArH),
7.22 (d, J = 8 Hz, 2H, ArH), 7.28–7.40 (m, 5H, ArH).
Butyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate
(14) Chromatographic purity (HPLC) 99.80 %; bp [ 300
8C; MS (ESI?) m/z = 348 [M?]; IR (KBr, cm-1): 2960,
2872, 1746, 1514, 1457, 1154, 847; 1H NMR (CDCl3,
400 MHz) d 0.76 (t, J = 7.2 Hz, 3H, CH3CH2), 0.82–0.89
(m, 9H, 39CH3), 1.27 (m, 2H, CH3CH2CH2), 1.40 (d,
J = 7.2 Hz, 3H, ArCHCH3), 1.50 (m, 2H, OCH2CH2CH2),
1.68–1.82 (m, 3H, CH(CH3)2/CHCH2CH3), 2.42 (d,
J = 7.2 Hz, ArCH2CH), 3.81–3.99 (m, 2H, OCH2CH2),
4.01–4.11 (m, 1H, ArCHCH3), 4.83 (m, 1H, OCHCO), 7.11
(d, J = 8 Hz, 2H, ArH), 7.22 (d, J = 8 Hz, 2H, ArH).
Cyclohexyl-2-(2-(4-isobutylphenyl)propanoyloxy)butano-
ate (10) Chromatographic purity (HPLC) 98.88 %;
bp [ 300 8C; MS (ESI?) m/z = 374 [M?]; IR (KBr,
cm-1): 2941, 2869, 1748, 1615, 1512, 1458, 1153, 844; 1H
NMR (CDCl3, 400 MHz) d 0.81–0.94 (m, 9H, 39CH3),
1.25–1.39 (m, 6H, 39CH2), 1.53 (m, 3H, ArCHCH3),
1.65–1.74 (m, 4H, 29CH2), 1.76–1.87 (m, 3H, CH(CH3)2/
CHCH2CH3), 2.43 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.79
(q, J = 7.2 Hz, 1H, ArCHCH3), 4.75–4.90 (m, 2H,
OCHCO/OCH(CH2)2), 7.09 (d, J = 8 Hz, 2H, ArH), 7.24
(d, J = 8 Hz, 2H, ArH), 7.28–7.40 (m, 5H, ArH).
Benzyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate
(15) Chromatographic purity (HPLC) 99.59 %; bp [
300 8C; MS (ESI?) m/z = 382 [M?]; IR (KBr, cm-1):
2955, 2870, 1740, 1608, 1514, 1456, 1383, 1197, 1107,
847, 750, 698; 1H NMR (CDCl3, 400 MHz) d 0.73 (t,
J = 7.2 Hz, 3H, CH3CH2), 0.82 (m, 6H, CH(CH3)2), 1.38
(t, J = 7.2 Hz, 3H, ArCHCH3), 1.66–1.81 (m, 3H,
CH(CH3)2, CH3CH2 CH), 2.41 (d, J = 7.2 Hz, 2H,
ArCH2CH), 3.83 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.90 (m,
1H, OCHCO), 5.09 (s, 2H, OCH2Ph), 7.09 (d, J = 8 Hz,
2H, ArH), 7.18 (d, J = 8 Hz, 2H, ArH), 7.27–7.38 (m, 5H,
ArH).
2-Ethylbutyl-2-(2-(4-isobutylphenyl)propanoyloxy)butano-
ate (11) Chromatographic purity (HPLC) 99.78 %; bp [
300 8C; MS (ESI?) m/z = 376 [M?]; IR (KBr, cm-1): 2966,
2877, 1748, 1514, 1460, 1151, 848; 1H NMR (CDCl3,
400 MHz) d 0.73–0.85 (m, 15H, 59CH3), 1.17–1.30 (m, 4H,
CH(CH2CH3)2), 1.46 (m, 1H, OCH2CH), 1.66–1.82 (m, 3H,
CH(CH3)2/CHCH2CH3), 1.40 (d, J = 7.2 Hz, 3H, ArCHCH3),
2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.79–3.95 (m, 2H, OCH2
CH), 3.96–4.06 (m, 1H, ArCHCH3), 4.85 (m, 1H, OCHCO),
7.11 (d, J = 8 Hz, 2H, ArH), 7.20 (d, J = 8 Hz, 2H, ArH).
Preparation of promoieties
Ethyl-2-chloro-3-oxobutanoate (2a)
2-Chloro-3-oxobutanoic acid (20 g, 0.15 mol) was treated
with ethanol (9.2 g, 0.2 mol) in the presence of toluene
(100 g, 1.08 mmol) as a solvent and PTSA (0.5 gm,
0.0026 mmol) as a catalyst. The reaction mass was main-
tained at reflux for 2 h. Reaction progress was monitored on
GC. After completion, reaction mass cooled to 30 °C and
washed twice with 200 mL 20 % sodium bicarbonate
solution in water followed by brine and water wash. Organic
layer dried over anhydrous sodium sulfate and distilled off
the solvent under reduced pressure and pure ethyl 2-chloro
3-oxobutanoate was collected as colorless oil.
2-Methoxyethyl-2-(2-(4-isobutylphenyl)propanoyloxy)but-
anoate (12) Chromatographic purity (HPLC) 99.81 %;
bp[ 300 8C; MS (ESI?) m/z = 350 [M?]; IR (KBr, cm-1):
1
2955, 2880, 1743, 1514, 1458, 1383, 1199, 1161, 848; H
NMR (CDCl3, 400 MHz) d 0.80–0.94 (m, 9H, 39CH3), 1.54
(m, 3H, ArCHCH3), 1.79–1.88 (m, 3H, CH(CH3)2/
CH3CH2CH), 2.43 (d, J = 7.2 Hz, ArCH2CH), 3.36 (s, 3H,
OCH3), 3.47–3.58 (m, 2H, CH2OCH3), 3.80 (m, 1H,
ArCHCH3), 4.21–4.30 (m, 2H, COOCH2), 4.90–4.95 (m, 1H,
OCHCO), 7.08–7.11 (m, 2H, ArH), 7.21–7.26 (m, 2H, ArH).
Chromatographic purity (HPLC) 97.73 %; bp 200 8C;
MS (ESI?) m/z = 164 [M?], 166 [M?2]; IR (KBr, cm-1):
Isopropyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate
(13) Chromatographic purity (HPLC) 99.97 %; bp 296
8C; MS (ESI?) m/z = 334 [M?]; IR (KBr, cm-1): 2978,
1
2985, 1732, 1645, 1621, 1257, 1032; H NMR (CDCl3,
400 MHz) d 1.34 (t, J = 7.6 Hz, 3H, CH2CH3), 2.39 (s,
123

Med Chem Res
3H, COCH3), 4.30 (q, J = 7.6 Hz, 2H, CH2CH3), 4.79 (s,
1H, CHCl).
4H (CH2CH3)2), 1.56 (septet, J = 6 Hz, 1H, CH(CH2)3),
3.84 (s, 2H, COCH2Br), 4.11 (d, J = 6 Hz, 2H, OCH2CH).
4-Bromobutyl acetate (2b)
2-Methoxyethyl-2-bromoacetate (2f)
4-Bromobutyric acid (20 g, 0.12 mol), methanol (9.5 g,
0.29 mol) in the presence of toluene (100 g, 1.08 mmol)
and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up
of reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 99.08 %; bp 211 8C;
MS (ESI?) m/z = 194 [M?], 196 [M?2]; IR (KBr, cm-1):
2963, 1739, 1438, 1237, 1042; 1H NMR (CDCl3,
400 MHz) d 1.81 (qu, J = 6.4 Hz, 2H, CH2CH2Br), 1.93
(qu, J = 6.4 Hz, 2H, CH2CH2O), 2.06 (s, 3H, COCH3),
3.44 (t, J = 6.4 Hz, 2H, CH2Br), 4.10 (t, J = 6.4 Hz, 2H,
COOCH2).
Bromoacetic acid (20 g, 0.14 mmol) 2-methoxyethanol
(9.16 g, 0.12 mmol) in the presence of toluene (100 g,
1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for
2 h. Work up of reaction carried out as per the procedure
given for 2a.
Chromatographic purity (HPLC) 98.74 %; bp 213 8C;
MS (ESI?) m/z = 198 [M?2]; IR (KBr, cm-1):2955,
2891, 2823, 1739, 1452, 1286, 1128, 1034; 1H NMR
(CDCl3, 400 MHz) d 3.40 (s, 3H, OCH3), 3.63 (t,
J = 4.4 Hz, 2H, OCH2CH2), 3.89 (s, 2H, COCH2Br), 4.33
(t, J = 4.4 Hz, 2H, COOCH2CH2).
Methyl-2-bromopropanoate (2c)
Isopropyl-2-bromoacetate (2g)
2-Bromopropionic acid (20 g, 0.13 mol), methanol (9.5 g,
0.29 mol) in the presence of toluene (100 g, 1.08 mmol)
and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up
of reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 97.72 %; bp 145 8C;
MS (ESI?) m/z = 166 [M?], 168 [M?2]; IR (KBr, cm-1):
Bromoacetic acid (20 g, 0.14 mmol), isopropanol (7.56 g,
0.12 mmol) in the presence of toluene (100 g, 1.08 mmol)
and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up
of reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 95.89 %; bp 274 8C;
MS (ESI?) m/z = 179 [M?]; IR (KBr, cm-1): 2968, 2881,
1
1
2968, 2878, 1736, 1458, 1270, 1156; H NMR (CDCl3,
1741, 1461, 1272, 1155; H NMR (CDCl3, 400 MHz) d
400 MHz) d 1.84 (d, J = 7.6 Hz, 3H, CH2CH3), 3.78 (s,
3H, OCH3), 4.40 (q, J = 7.6 Hz, 1H, CHCH3).
1.28 (d, J = 6.4 Hz, 6H, CH(CH3)2), 3.80 (s, 2H,
COCH2Br), 5.06 (septet, J = 6.4 Hz, 1H, CH(CH3)2).
Cyclohexyl-2-bromoacetate (2d)
Butyl-2-bromoacetate (2h)
Bromoacetic acid (20 g, 0.14 mmol), cyclohexanol (12.9 g,
0.12 mmol) in the presence of toluene (100 g, 1.08 mmol)
and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of
reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 98.80 %; bp 242 8C;
MS (ESI?) m/z = 221 [M?]; IR (KBr, cm-1):2965, 2877,
Bromoacetic acid (20 g, 0.14 mmol),n-bromobutanol(9.60 g,
0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and
PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of
reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 97.30 %; bp 92 8C; MS
(ESI?) m/z = 195 [M?]; IR (KBr, cm-1): 2937, 2875, 1738,
1
1
1736, 1451, 1233, 1163; H NMR (CDCl3, 400 MHz) d
1458, 1283, 1170; H NMR (CDCl3, 400 MHz) d 0.94 (t,
1.42–1.57 (m, 6H, 29CH2), 1.72–1.88 (m, 4H, CH(CH2)2),
3.81 (s, 2H, COCH2Br), 4.82 (m, 1H, CH(CH2)2).
J = 7.6 Hz, 3H, CH2CH3), 1.40 (sextet, J = 7.6 Hz, 2H,
CH2CH2CH3), 1.65 (qu, J = 7.6 Hz, 2H, CH2CH2CH3),
3.83 (s, 2H, COCH2Br), 4.18 (t, J = 6.8 Hz, 2H,
OCH2CH2).
2-Ethylbutyl-2-bromoacetate (2e)
Bromoacetic acid (20 g, 0.14 mmol), 2-ethyl butanol (12.86 g,
0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and
PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of
reaction carried out as per the procedure given for 2a.
Benzyl-2-bromoacetate (2i)
Bromoacetic acid (20 g, 0.14 mmol), Benzyl alcohol (13.72 g,
0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and
PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of
reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 98.60 %; bp 261 8C;
MS (ESI?) m/z = 223 [M?]; IR (KBr, cm-1): 2969, 2876,
1
1742, 1458, 1265, 1162; H NMR (CDCl3, 400 MHz) d
0.90 (t, J = 7.4 Hz, 6H, (CH2CH3)2), 1.37 (qu, J = 7.4 Hz,
Chromatographic purity (HPLC) 96.82 %; bp 287 8C;
MS (ESI?) m/z = 228 [M?]; IR (KBr, cm-1): 3033, 2960,
123

Med Chem Res
1738, 1455, 1278, 1156, 971; 1H NMR (CDCl3, 400 MHz)
d 3.84 (s, 2H, COCH2Br), 5.18 (s, 2H, OCH2Ph), 7.35 (m,
5H, ArH).
and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of
reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 98.19 %; bp 213 8C;
MS (ESI?) m/z = 208 [M?]; IR (KBr, cm-1): 2981, 2939,
1
2-Bromo-butyric acid cyclohexyl ester (2j)
2880, 1738, 1458, 1272, 1161, 1105; H NMR (CDCl3,
400 MHz) d 1.02 (t, J = 7.6 Hz, 3H, CH3CH2), 1.26 (d,
J = 6.4 Hz, 6H, CH(CH3)2), 1.96–2.13 (m, 2H,
CH2CHBr), 4.12 (t, J = 7.6 Hz, 1H, CH2CHBr), 5.06
(sept, J = 6.4 Hz, 1H, CH(CH3)2).
Bromobutyric acid (20 g, 0.14 mmol), cyclohexanol (10.70 g,
0.10 mmol) in the presence of toluene (100 g, 1.08 mmol) and
PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of
reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 99.00 %; bp 280 8C;
MS (ESI?) m/z = 249 [M?]; IR (KBr, cm-1): 2939, 2860,
2-Bromo-butyric acid butyl ester (2n)
1
1732, 1455, 1262, 1161; H NMR (CDCl3, 400 MHz) d
2-Bromobutyric acid (20 g, 0.11 mmol), n-butanol (7.3 g,
0.09 mmol) in the presence of toluene (100 g, 1.08 mmol)
and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up
of reaction carried out as per the procedure given for 2a.
Chromatographic purity (HPLC) 98.80 %; bp 255 8C; MS
(ESI?) m/z = 223 [M?]; IR (KBr, cm-1): 2963, 2877, 1740,
1.04 (t, J = 7.2 Hz, 3H, CH3CH2), 1.27–1.56 (m, 6H,
39CH2), 1.70-1.88 (m, 4H, 29CH2), 1.85–2.15 (m, 2H,
CH2CHBr), 4.14 (t, J = 7.2 Hz, 1H, COCHBr), 4.83 (m,
1H, COOCH).
1
2-Bromo-butyric acid 2-ethyl-butyl ester (2k)
1458, 1271, 1157; H NMR (CDCl3, 400 MHz) d 0.94 (t,
J = 7.6 Hz, 3H, CH3CH2CH2), 1.03 (t, J = 7.2 Hz, 3H,
CH3CH2CHBr), 1.40 (sextet, J = 7.6 Hz, 2H, CH3CH2CH2),
1.66 (qu, J = 7.6 Hz, 2H, OCH2CH2), 1.95–2.16 (m, 2H,
CH2CHBr), 4.13–4.22 (m, 3H, OCH2, CH2CHBr).
2-Bromobutyric acid (20 g, 0.11 mmol), 2-ethyl butanol
(10.11 g, 0.09 mmol) in the presence of toluene (100 g,
1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for
2 h. Work up of reaction carried out as per the procedure
given for 2a.
2-Bromo-butyric acid benzyl ester (2o)
Chromatographic purity (HPLC) 98.88 %; bp 292 8C;
MS (ESI?) m/z = 251 [M?]; IR (KBr, cm-1): 2965, 2878,
2-Bromobutyric acid (20 g, 0.11 mmol), benzyl alcohol
(10.72 g, 0.09 mmol) in the presence of toluene (100 g,
1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for
2 h. Work up of reaction carried out as per the procedure
given for 2a.
1
1741, 1460, 1268, 1156; H NMR (CDCl3, 400 MHz) d
0.90 (t, J = 7.6 Hz, 6H, 29CH3CH2), 1.02 (t, J = 7.6 Hz,
3H, CH3CH2), 1.38 (qu, J = 7.6 Hz, 4H, 29CH2CH3),
1.56 (sept, J = 7.6 Hz, 1H, CH(CH3)2), 1.97–2.14 (m, 2H,
CH2CHBr), 4.06–4.18 (m, 3H, COOCH2, CH2CHBr).
Chromatographic purity (HPLC) 98.45 %; bp 295 8C;
MS (ESI?) m/z = 256 [M?]; IR (KBr, cm-1): 2973, 1741,
1
1455, 1266, 1150; H NMR (CDCl3, 400 MHz) d 1.00 (t,
2-Bromo-butyric acid 2-methoxy-ethyl ester (2l)
J = 7.2 Hz, 3H, CH3CH2), 1.97–2.15 (m, 2H, CH2CHBr),
4.20 (t, J = 7.2 Hz, 1H, CH2CHBr), 5.20 (s, 2H,
OCH2Ph), 7.36 (m, 5H, ArH).
2-Bromobutyric acid (20 g, 0.11 mmol), 2-methoxy etha-
nol (7.5 g, 0.09 mol) in the presence of toluene (100 g,
1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for
2 h. Work up of reaction carried out as per the procedure
given for 2a.
Anti-inflammatory activity evaluation in animal models
Oral acute toxicity study
Chromatographic purity (HPLC) 98.46 %; bp 181 8C;
MS (ESI?) m/z = 225 [M?]; IR (KBr, cm-1): 2975, 2881,
Acute toxicity of various prodrugs was carried out in Swiss
albino mice (20–25 g) as per the OECD guidelines (2006).
Different doses of various prodrugs were administered to
groups (n = 3) up to 2,000 mg/kg orally and animals were
observed for a period of 72 h for behavioral changes, toxic
reactions, and mortality if any.
1
1743, 1457, 1262, 1158; H NMR (CDCl3, 400 MHz) d
1.04 (t, J = 7.2 Hz, 3H, CH3CH2), 1.96–2.15 (m, 2H,
CH2CHBr), 3.39 (s, 3H, OCH3), 3.61 (t, J = 4.8 Hz, 2H,
CH2CH2O), 4.21 (t, J = 7.2 Hz, 1H, CH2CHBr), 4.32 (t,
J = 4.8 Hz, 2H, COOCH2CH2).
2-Bromo-butyric acid isopropyl ester (2m)
Carrageenan-induced hind paw edema in rats
2-Bromobutyric acid (20 g, 0.11 mmol), Isopropanol (5.94 g,
0.09 mmol) in the presence of toluene (100 g, 1.08 mmol)
Dose response study Rats were divided into various groups
(n = 6) and allowed to free access to water ad libitum.
123

Med Chem Res
In vivo metabolic stability study (plasma drug
concentration determination)
Different groups of rats administered with ibuprofen
(100 mg/kg, b.w.) and various prodrugs (100 mg/kg, v)
orally. One group of rats served as a control and administered
with gum acacia (1 %, w/v; 10 mL/kg, b.w., p.o.). One hour
after the drug administration, to all rats, hind paw edema was
induced by the method of winter et al. (1962) by injecting
0.1 mL of 1 % (w/v) solution of carrageenan subcutaneously
into the subplanter region of hind paw.
Metabolic stability is an important property of drug candi-
dates, as it affects parameters such as plasma clearance,
half-life, and bioavailability. A successful prodrug candi-
date is expected to undergo rapid, complete conversion to
parent compound in the plasma or microsomes within 1–3 h
for its desired pharmacological action. The metabolic sta-
bility study was carried out in the following method. The
male Sprague-Dawley rats were used to compare the bio-
availability of prodrugs (2, 6, 9, 10, 12, and 14) with that of
ibuprofen (1a) following oral administration. Overnight
fasted rats were divided into various groups (n = 6), and
they were administered prodrugs (2, 6, 9, 10, 12, and 14) at
the dose of 100 mg/kg, orally. Blood sample from each rats
were withdrawn through retro orbital plexus in eppendorf
tube (containing EDTA 5 % as anticoagulant) at various
time intervals viz; 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h after the
drug administration and centrifuged at 5000 rpm for 10 min
for plasma separation. To 200 lL of plasma, 200 lL of
acetonitrile solution was added and vortexed for 2 min for
the precipitation of protein which was separated by centri-
fugation. The clear supernatant obtained and used for
determination of concentration by HPLC technique. The
area under curve (AUC) is calculated by
Time course study The hind paw edema volume was
measured by volume displacement method using plethys-
mometer (Orchid scientific; 112, India) by immersing the
paw till the level lateral malleolus at various time intervals
(1, 3 and 6 h) after carrageenan injection.
Cotton pellets-induced granuloma formation in rats
Two sterilised cotton pellets (10 mg) were implanted on
ventral regions of rats, procedure described by winter and
porter (Winter and Porter, 1957) and divided into various
groups (6 rats/group). Different groups of rats administered
with ibuprofen (100 mg/kg) and various prodrugs (100 mg/
kg) orally for a duration of 8 days. Control group received
gum acacia (2 %, 10 mL/kg, p.o.). On the day 9th, rats
were sacrificed with excess ether anesthesia. The cotton
pellets were removed and freed from extraneous tissue and
used for granular tissue formation by recording wet
(immediately) and dry weight of pellets. The granular tis-
sue formation was studied by drying cotton pellets at 55 °C
for 6 h or till the weight of pellets remains constant. The
dry weight was calculated after deducting cotton pellet
weight and taken as a granular tissue formation.
C1
AUC ¼ C0 þ
ꢁ Time1ꢂ0
2
where, C0 represents the peak area at time t0 and C1 rep-
resents the peak area at time t1.
Microsomal stability
Materials Nicotinamide adenine dinucleotide phosphate
(NADPH) (Sigma, lot no. N 6674); acetonitrile (HPLC
grade, Merck, India); Tris–HCl (S.D.Fine-Chem, India); rat
liver microsomes (isolated in pharmacology laboratory);
prodrugs; and standard ibuprofen.
Measurement of ulcer index
Ulcer index were evaluated in 2 experimental conditions
(a) For the normal 24 h fasted rats were taken and were
administered with test compounds orally at 400 mg/kg and
24 h after the drug administration, animals were scarified
and stomach were isolated and observed for number of
ulcers, (b) in cotton pellets-induced granuloma in rats-
Immediately after cotton removal in above experiment,
stomach was dissected out, incised along the greater cur-
vature and mucosa were rinsed with cold normal saline
solution (pH 7.2) to remove blood contaminant if any. The
tissue were kept overnight in 10 % formalin solution. Next
day, ulcers were examined under the 109 lens using
method described in the literature (Ganguly and Bhatnagar,
1973) on the scale [0 = no ulcer; 1 = hemorrhagic patch;
2 = ulcer \2 mm; 3 = ulcer [2 mm or \4 mm; 4 =
ulcer [4 mm]. The ulcer index was calculated as mean
SEM of 6 rats in each group.
Requirement Required items were as follows: rat liver
microsomes (10 mg/mL protein concentration), NADPH
(10 mM solution), Tris–HCl buffer (pH 7.4), Eppendorf
tubes, and solutions of various prodrugs of ibuprofen
(5 mM).
Experimental conditions Incubation period, 60 min;
incubation conditions, 37 °C with 60 rpm shaking; protein
precipitation solvent, acetonitrile.
Procedure In 50 lL of rat liver microsomes, 100 lL
Tris–HCl buffer was added, with 50 lL of NADPH solu-
tion(10 mM), and were mixed thoroughly and vortexed
123

Med Chem Res
subsequently for 10 s. To this mixture, 5 lL prodrug
solution (5 mM) was injected and vortexed well. The
75 lL sample was withdrawn immediately and transferred
to the centrifuge tube containing 75 lL ice cold acetoni-
trile, vortexed, and centrifuged at 14,000 rpm for 10 min.
Aliquots of the supernatant was separated and used for
HPLC analysis. Then the assay mixture was incubated in a
water bath at 37 °C for 60 min. At specific time points (30
and 60 min), the75 lL assay mixture was withdrawn and
vortexed in equal volume of cold acetonitrile and centri-
fuged again at 14,000 rpm for 10 min. Aliquots of the
supernatant were separated and used for analysis by HPLC.
The percentage of prodrug remaining in rat microsomes
was calculated by:
(Scheme 1). The structures of all prodrugs were established
by IR, 1H NMR, mass spectrometry, and their purity in
excess of 99 % was confirmed by HPLC analysis.
The syntheses of promoieties 2a–c were outlined in
Scheme 2. The promoieties 2d–i were obtained by reflux-
ing 2-bromoacetic acid with respective alcohols in the
presence of toluene and catalytic amount of PTSA
(Scheme 3). Similarly, promoieties 2j–o were obtained by
refluxing 2-bromobutanoic acid with respective alcohols in
the presence of toluene and catalytic amount of PTSA
(Scheme 4). The structures of all promoieties were estab-
lished by IR, 1H NMR, and mass spectrometry, and their
purity in excess of 97 % was confirmed by HPLC analysis.
PeakareaatrespectivetimeðminÞ ꢁ 100
Prodrug remaining ð%Þ ¼
Peak area at 0 min
Aqueous solubility
Pharmacological evaluation
The solubility of prodrugs 2, 6, 9, 10, 12, and 14 was
determined using boric acid buffer (pH 9.0) at 40 °C,
0.2 mM phosphate buffer (pH 7.4), 0.1 mM phosphate
buffer (pH 5.0), 0.1 mM citric acid buffer (pH 3.0), and
0.2 M hydrochloric acid (pH 1.0). The various prodrugs of
ibuprofen (5 mg) in buffer solution (10 mL) were incu-
bated at 40 °C for 4 h at 400 rpm on a mechanical shaker.
The solution was filtered through 0.20 lM membrane filter
and the filtrate was analyzed quantitatively by HPLC at a
wavelength of 254 nM for its solubility and hydrolysis.
The therapeutic use of NSAIDs is often restricted by the
necessity to deliver the drug to specific sites of target organ
or tissue. The use of NSAIDs is also limited by their irritant
side effects on the gastro-enteric mucous and by their poor
water solubility. A series of ester prodrugs 1–15 of ibu-
profen 1a was synthesized with the aim of minimizing
these side effects and to enhance the oral bioavailability.
Acute toxicity study
There were no significant behavioral changes observed
with all the employed doses of prodrugs (1–15). To all the
mice, neither toxic reaction nor mortality observed.
Therefore, 2,000 mg/kg, body weight (b.w.), per oral (p.o.)
dose was considered as maximum tolerated dose. Based on
acute toxicity study, we have selected 100 mg/kg for anti-
inflammatory activity evaluation of various prodrugs
(1–15).
Metabolic stability
For the HPLC analysis system; water alliance 2695-sepa-
ration module with 2996 PDA detector, C18 Column, ODS
3 v, 250 mm 9 4.6 mm, 5 lM, and mobile phase was
A-10 mM potassium dihydrogen phosphate, wavelength,
265 nM and for B-acetonitrile at various ratios (90:10,
30:70, 10:90 and 70:30).
Anti-inflammatory activities evaluation
Results and discussion
In a dose response study in carrageenan edema, there was
significant (p \ 0.01) increase in paw volume of carra-
geenan control rats when compared with normal rats. All
prodrugs significantly inhibited the edema formation
(p \ 0.01) than the parent drug ibuprofen. However, the
extent of edema inhibition in rats treated with 2, 6, 9, 10,
12, and 14 was much greater at 3 and 6 h than other pro-
drugs when compared to carrageenan control rats
(Table 1). Inhibition of paw edema formation at 6 h is
Chemistry
A new series of ibuprofen ester prodrugs 1–15 (Fig. 1) were
readily prepared in good yields by the condensation of
ibuprofen 1a with appropriate promoieties 2a–o (Fig. 2), in
the presence of potassium carbonate in dimethyl acetamide
(DMAc) at room temperature under nitrogen atmosphere
123

Med Chem Res
Fig. 1 Chemical structures of
ibuprofen (1a) and produrgs
1–15
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
Ibuprofen (1a)
1
3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
4
5
7
O
O
O
6
O
O
9
8
O
O
10
11
13
15
O
O
O
12
O
O
14
greater due to the inhibition of prostaglandins, which is
known to be released at 4–6 h after the carrageenan
injection (Crunckhon and Meacock, 1971). Prodrugs elic-
ited greater paw edema inhibition (2, 6, 9, 10, 12, and 14)
was taken for further evaluation.
found with prodrugs 2, 12 and 14 when compared with
control rats. Ibuprofen treatment also showed similar
inhibitory effects on both the phases of inflammation
(Table 2).
In cotton pellet-induced granuloma in rats, all the
selected prodrugs 2, 6, 9, 10, 12, and 14 were significantly
inhibited (p \ 0.01) both the exuhdatory and granulatory
phases of inflammation than the parent drug ibuprofen (1a).
However, greater degree inhibition of these phases was
Ulcerogenic assay
Ulcer index in rats during cotton pellet-induced granu-
loma In this model, lesser degree of ulcers was observed in
123

Med Chem Res
Fig. 2 Chemical structures of
ethyl-2-chloro-3-oxobutanoate
(2a), 4-bromobutyl acetate (2b),
methyl-2-bromopropanoate
(2c), cyclohexyl-2-
bromoacetate (2d),
2-ethylbutyl-2-bromoacetate
(2e), 2-methoxyethyl-2-
O
O
O
Br
Cl
Br
O
O
O
O
2a
2b
2c
O
O
O
bromoacetate (2f), isopropyl-2-
bromoacetate (2g), butyl-2-
bromoacetate (2h), benzyl-2-
bromoacetate (2i), 2-bromo-
butyric acid cyclohexyl ester
(2j), 2-bromo-butyric acid
2-ethyl-butyl ester (2k),
Br
Br
Br
Br
Br
O
Br
Br
Br
Br
O
2e
O
O
O
O
O
2d
2f
O
O
O
Br
Br
Br
O
O
2-bromo-butyric acid
2-methoxy-ethyl ester (2l),
2-bromo-butyric acid isopropyl
ester (2m), 2-bromo-butyric
acid butyl ester (2n), 2-bromo-
butyric acid benzyl ester (2o)
2g
2h
2i
O
O
O
O
O
O
2j
2k
2l
O
O
O
O
O
2o
2m
2n
Scheme 1 Synthesis of
ibuprofen ester prodrugs 1–15.
Reagents and conditions:
(i) promoieties 2a–o, K2CO3,
DMAc, rt
CH3
CH3
O
OH
CH3
R
CH3
i
O
O
H3C
H3C
Ibuprofen (1a)
Prodrugs of Ibuprofen (1-15)
(1) R = CH(COCH3)COOCH2CH3
(2) R = CH2CH2CH2CH2OCOCH3
(3) R = CH(CH3)COOCH3
(8) R = CH2COO(CH2)3CH3
(9) R = CH2COOCH2Ph
(10) R = CH(CH2CH3)COOC6H11
(11) R = CH(CH2CH3)COOCH2CH(CH2CH3)2
(12) R = CH(CH2CH3)COOCH2CH2OCH3
(13) R = CH(CH2CH3)COOCH(CH3)2
(14) R = CH(CH2CH3)COO(CH2)3CH3
(15) R = CH(CH2CH3)COOCH2Ph
(4) R = CH2COOC6H11
(5) R = CH2COOCH2CH(CH2CH3)2
(6) R = CH2COOCH2CH2OCH3
(7) R = CH2COOCH(CH3)2
rats treated with prodrugs 2, 6, 9, 10, 12, and 14 when
compared to standard ibuprofen (Table 3). Administration
of ibuprofen 1a and prodrugs 2, 6, 9, 10, 12 and 14 (100 mg/
kg, b.w., p.o.) daily for 7 days in rats implanted with cotton
pellet-induced ulcers. As the ibuprofen was found to produce
irritation and ulceration of the GI mucosa, has arisen in
clinical trials (Ashraf et al., 1999; Kean and Buchanan,
2005). The GI ulceration in both the experimental conditions
may be due to the presence of carboxylic acid functional
group present in ibuprofen, inhibition of protective prosta-
glandins and inhibition of mucus production. Rats treated
with standard drug ibuprofen 1a had ulcer index of about
123

Med Chem Res
Scheme 2 Reagents and
conditions: (a) PTSA, toluene,
reflux, 2 h
O
O
O
Cl
O
a
a
+
+
OH
OH
OH
OH
OH
O
Cl
2a
O
O
Br
Br
O
2b
O
O
a
Br
CH3-OH
+
O
Br
2c
Scheme 3 Reagents and
conditions: (a) PTSA, toluene,
reflux, 2 h
O
O
Br
O
Br
O
2d
OH
2i
HO
Ph
a
a
O
OH
O
O
Br
HO
O
Br
Br
O
HO
a
a
2e
2h
O
HO
a
a
OH
O
O
Br
O
Br
O
O
2g
2f
6 mm lesion and found to be much greater than all the syn-
thesized prodrugs (Fig. 3). Rats treated with 2 (0.1 ulcer), 12
(0.1 ulcer) and 14 (0.1 ulcer) found to developed no
ulcerogenic lesions as same that of ibuprofen 1a (6 ulcer).
These experimental findings revealed that the presence of
functional groups like, 4-bromobutyl acetate, 2-methoxy
ethyl-2-bromobutanoate, and butyl-2-bromo-butanoate on
carboxylic acid to 2, 12, and 14 respectively. However, other
prodrugs, viz. 6, 9, and 10 also showed lesser degree of ulcer
formations but comparatively greater than 2, 12, and 14.
Therefore, 2, 12, and 14 are most potent anti-inflammatory
prodrugs with no or less ulcerogenic potential as compared to
parent ibuprofen.
400 mg/kg showed high incidence of ulcers when compared
with normal group; however, the lesser degree of ulcer for-
mation was observed in test prodrugs 2, 6, 9, 10, 12, and 14 as
compared to standard ibuprofen 1a (Table 4). Rats treated
with standard drug ibuprofen 1a had ulcer index of about 10.2
and found to be much greater than the entire group of tested
prodrugs (Fig. 4). This gives clear indication that ibuprofen is
highly ulcerogenic than other ester prodrugs. Furthermore,
compounds 2, 12, and 14 were found to produce least ulcer in
normal rats at 400 mg/kg, indicating significant anti-inflam-
matory activity with no ulcer formation.
The present experimental findings demonstrated that
4-bromobutyl acetate, 2-methoxy ethyl-2-bromobutanoate,
and butyl-2-bromo-butanoate ester prodrugs of ibuprofen
protected the gastric mucosal damage induced by ibuprofen.
In this respect, our findings shed new chemical requisite to
Ulcer index in normal rats Experimental findings
demonstrated that rats administered with ibuprofen 1a at
123

Med Chem Res
Scheme 4 Reagents and
conditions: (a) PTSA, toluene,
reflux, 2 h
O
O
Br
Br
O
O
OH
2o
2j
HO
Ph
a
a
O
OH
O
O
Br
HO
Br
O
O
OH
a
a
Br
2k
2n
O
HO
a
a
OH
O
O
Br
Br
O
O
O
2m
2l
design new agents of ibuprofen with no ulcerogenic effects
on GI system.
Table 1 Effects of various prodrugs 1–15 on carrageenan-induced
paw edema in rats
Treatment and dose Paw volume (mL) (Mean SEM) at various
(100 mg/kg p.o.)
time interval
In vivo metabolic stability study (plasma drug
concentration determination)
1 h
3 h
6 h
Normal group
0.40 0.04 0.35 0.04
0.38 0.06
The hydrolysis studies were also studied in male Wistar rats.
All selected ester prodrugs (2, 6, 9, 10, 12, and 14) were
quantitatively hydrolyzed to the parent drug ibuprofen by
enzymatic conversion in vivo; their respective release
characteristics were observed (Fig. 5). The Cmax (maximum
concentration) of standard ibuprofen 1a was 91.2 mg/L and
(AUC)0–t was 239, whereas Cmax of 2, 6, 9, 10, 12, and 14
were 70.1, 73.2, 78.0, 80.2, 92.3, 115.2 mg/L respectively
with AUC0–t of 2, 6, 9, 10, 12, and 14 were 188, 244, 241,
237, 192 and 335 respectively (Table 5).
Carrageenan control 0.94 0.05# 1.17 0.10# 1.69 0.08##
Ibuprofen (1a)
0.84 0.05 1.00 0.07
1.20 0.04 0.96 0.10
1.03 0.10 0.98 0.10
1.12 0.06 0.97 0.05
1.29 0.10 1.11 0.10
0.88 0.10 0.98 0.10
1.03 0.10 1.00 0.10
1.11 0.04 1.10 0.03
1.10 0.04 1.10 0.03
0.73 0.05**
1.26 0.10
0.84 0.10**
1.20 1.10
1.01 0.10*
1.19 0.10
0.89 0.10**
1.18 0.20
1.18 0.20
1
2
3
4
5
6
7
Bioavailability was measured by calculating the AUC of
the drug concentration time profile. The plasma concentra-
tion of compound 14 was found to maximum as compared to
parent drug ibuprofen indicating improved bioavailability.
Prodrug 10 showed slow released rate than other compounds
which indicate that slow enzymatic conversion of prodrug
moiety into parent compound. Similarly, compound 12 also
showed same characteristic release rate in vivo with Cmax of
92.3 with higher bioavailability compared to all other pro-
drugs except 14 indicate fast release of prodrug into parent
ibuprofen for its pharmacological action.
8
9
1.26 0.15 0.68 0.03** 0.75 0.10**
0.76 0.10 0.95 0.10* 0.82 0.10**
0.93 0.10 0.89 0.10* 1.00 0.10*
10
11
12
13
14
15
1.04 0.10 0.93 0.1*
0.22 0.10**
1.09 0.10 0.85 0.05* 0.98 0.03*
0.80 0.10 1.05 0.10
1.03 0.05 0.96 0.10
0.81 0.01**
1.00 0.05*
#
##
p \ 0.05; p \ 0.01 when compared with normal control group.
** p \ 0.01; * p \ 0.05 when compared with carrageenan control
group
123

Med Chem Res
Table 2 Effects of various prodrugs on cotton pellet-induced gran-
uloma in rats
Table 4 Ulcer index of various prodrugs in normal rats
Treatment and dose
(400 mg/kg p.o.)
Number of ulcer
(Mean SEM)
Ulcer index
(Mean SEM)
Treatment and dose (100 mg/kg p.o.) Weight (mg) (Mean SEM)
Wet
Dry
Normal control
0.00 0.0
0.10 0.01
Ibuprofen (1a)
7.2 0.20##
0.2 0.03**
0.6 0.01**
0.5 0.04**
0.7 0.01**
0.2 0.01**
0.02 0.01
10.3 1.10##
0.20 0.02**
0.80 0.01**
0.80 0.01**
1.20 0.09**
0.20 0.01**
0.20 0.01**
Normal control
256 11.2
153 9.9
2
Ibuprofen (1a)
148 11.5**
174 8.5**
170 13.8**
165 13.2**
194 4.2*
76 4.9
104 3.9
90.4 5.9
92.8 4.6
122.4 3.9
77.4 7.2
79 1.9
6
2
9
6
10
12
14
9
10
12
14
178 5.08**
161 3.9**
#
##
p \ 0.05; p \ 0.01 when compared with normal control group;
** p \ 0.01, * p \ 0.05 when compared with ibuprofen group
** p \ 0.01; * p \ 0.05 when compared with normal control group
#
##
p \ 0.05; p \ 0.01 when compared with normal control group
** p \ 0.01; * p \ 0.05 when compared with vehicle treated control
group
Table 3 Ulcer index of various prodrugs in cotton pellets-induced
granuloma in rats
Treatment and dose
(400 mg/kg p.o.)
Number of ulcer
(Mean SEM)
Ulcer index
(Mean SEM)
Normal control
0.00 0.0
0.10 0.01
6.0 0.30##
0.1 0.02**
1.0 0.01**
1.0 0.01**
1.1 0.07**
0.1 0.02**
0.1 0.01**
Ibuprofen (1a)
5.1 0.20##
0.2 0.03**
0.7 0.01**
0.8 0.04**
0.9 0.01**
0.2 0.01**
0.2 0.01**
2
6
Fig. 4 Ulcer index of various prodrugs in normal rats
9
10
12
14
#
##
p \ 0.05; p \ 0.01 when compared with normal control group
** p \ 0.01; * p \ 0.05 when compared with ibuprofen group
Fig. 5 Plasma concentration of ibuprofen and prodrugs at various
time intervals
Physicochemical properties
Fig. 3 Ulcer index of various prodrugs in cotton pellets-induced
granuloma in rats
Aqueous solubility All the drugs administered by oral
route are supposed to stable at various pH environments
encountered in the GI to convert the intact prodrug to the
systemic circulation and reach to site of action. Prodrugs of
ibuprofen candidate (2, 6, 9, 10, 12, and 14) were evaluated
for solubility and stability in a series of buffer solutions
In vivo metabolic stability study (microsomal stability)
From experimental findings it is found that ester prodrugs of
ibuprofen were highly metabolized however, standard ibu-
profen was stable up to 60 min in liver microsomes (Table 6).
123

Med Chem Res
Table 5 Area under curve and plasma concentration of various test
compounds
Table 8 Partition coefficients of ibuprofen and prodrugs (2, 6, 9, 10,
12, 14) in buffer solutions at 20 °C and pH 4 and 7.4
Compound
Area under
curve (AUC)
Plasma concentration
(mg/lit)
Prodrug
Partition coefficients of prodrugs at various pH
3.0
7.4
Ibuprofen (1a)
239
188
244
241
237
192
335
91.2
70.1
73.2
78.0
80.2
92.3
115.2
Ibuprofen (1a)
3.75
3.68
4.12
4.09
4.76
3.91
4.05
2.45
4.11
3.49
4.21
4.57
4.02
4.25
2
2
6
6
9
9
10
12
14
10
12
14
Results reported are the average of three experiments (n = 3)
Table 6 Metabolic stability of various prodrugs of ibuprofen
Partition coefficient (log P) The partition coefficients
(log P) of ibuprofen 1a and prodrugs 2, 6, 9, 10, 12, 14
were determined by HPLC method in octanol-buffer sys-
tem. Partition coefficient values for all prodrugs were
found to be higher than ibuprofen at pH 7.4. Results of log
P at pH 3.0 and pH 7.4 are summarized in Table 8. Higher
log P values of all prodrugs at pH 7.4, indicated that pro-
drugs are more lipophilic than the parent drug.
Compound
Metabolic stability in rat liver microsomes at various
time intervals (min)
0
30
60
90
Ibuprofen (1a) 100
92.1
8.1
9.2
1
79.2
0
58.3
0
2
100
100
100
100
100
100
6
0
0
9
0
0
10
12
14
1.2
2.1
0
0
0
0
0
Conclusions
0
0
The percentage of prodrugs remaining in rat microsomes was calcu-
lated by Prodrug remaining (%) = peak area at respective time (min)/
peak area at 0 min 9 100
A series of novel ester prodrugs of ibuprofen have been
synthesized and evaluated in vivo fortheir anti-inflammatory
potential using carrageenan-induced rat hind paw edema and
cotton pellet-induced granuloma models inflammation along
with non-ulcerogenic potential. These prodrugs emerged out
as anti-inflammatory prodrugs with lesser degree of ulcero-
genic potential than the parent drug ibuprofen. Ulcer index
(UI) studies revealed that the ibuprofen prodrugs 2, 12, and
14 were substantially less ulcerogenic in both normal rats
(UI = about 0.1) and inflammation-induced rats (cotton
ranging from pH 1 to 9. All the ibuprofen prodrugs were
found to be stable and sparingly soluble in buffer solutions
(below detection limit). Aqueous solubility of 9, 10, 12,
and 14 was improved; however, it is lower than parent
ibuprofen (Table 7). Furthermore, the degradation of pro-
drugs 9, 10, 12, and 14 indicted a substantially higher
chemical stability in an aqueous buffer solution.
Table 7 Aqueous solubility, stability of ibuprofen and various prodrugs (2, 6, 9, 10, 12, 14) in buffer solutions at 40 °C for 4 h
Prodrug
Hydrolysis to parent drug (lg/mL) at various pH
Solubility (lg/mL) at various pH
1.0
3.0
5.0
7.4
9.0
1.0
3.0
5.0
7.4
9.0
Ibuprofen (1a)
–
–
–
–
–
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
2
0.0735
0.0936
0.2253
0.2361
0.2990
0.0323
0.0122
0.0975
0.0255
0.1258
0.2415
0.0153
0.1250
0.2965
0.2600
0.0556
0.0115
0.0026
0.1256
0.1252
0.2510
0.0255
0.0241
0.2990
0.0090
0.2521
0.2987
0.3565
0.3325
0.1988
6
9
10
12
14
Limit of detection is estimated by HPLC method; ibuprofen (1a) (0.0081 lg/mL), 2 (0.0162 lg/mL), 6 (0.0615 lg/mL), 9 (0.0071 lg/mL), 10
(0.0121 lg/mL), 12 (0.0072 lg/mL), 14 (0.0411 lg/mL)
BDL below detection limit
123

Med Chem Res
Ganguly AK, Bhatnagar OP (1973) Effect of bilateral adrenalactomy
on production of resistant ulcers in the stomach of albino rats.
Can J Physiol Pharmacol 51:748–750
Halen PK, Murumkar PR, Giridhar R, Yadav MR (2009) Prodrug
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Huang Z, Velazquez CA, Abdellatif KRA, Chowdhury MA, Reisz JA,
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pellets-induced granuloma; UI = about 0.2) than the parent
ibuprofen 1a (UI = 6 and 10.3 respectively). Metabolic
stability study showed that prodrug 2, 12 and 14 were rapidly
transformed enzymatically into parent drug ibuprofen in
both rat liver microsomes and rat plasma. Based on these
experimental observations, prodrug 12 and 14 are potent and
safe anti-inflammatory and analgesic agents with least GI
irritation and ulceration therefore, can be preferred over
parent drug ibuprofen for the treatment of inflammation
Kawail S, Kojima F, Kusunoki N (2005) Recent advances in non-
steroidal anti-inflammatory drugs. Allergol Int 54:209–215
Kean WF, Buchanan WW (2005) The use of NSAIDs in rheumatic
disorders 2005: a global perspective. Inflammopharmacol 13:343–370
Kim H, Jeon H, Kong H, Yang Y, Choi B, Kim YM, Neckers L, Jung
YA (2006) Molecular mechanism for the anti-inflammatory
effect of taurine-conjugated 5-aminosalicylic acid in inflamed
colon. Mol Pharmacol 69:1405–1412
Acknowledgments The authors also gratefully acknowledge the
support extended by Mr. Ashok Hiremath, Managing Director, Astec
Lifesciences Ltd. Authors are also thankful to Dr. S. R. Naik and Dr.
S.B. Bhise, Sinhgad Institute of Pharmaceutical Sciences, Lonavala
for providing necessary facility for pharmacological experimentation.
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